Summary The devastating motor neuron diseases Amyotrophic Lateral Sclerosis and Spinal Muscular Atrophy cause differential degeneration of subsets of motor neurons. Humans possess hundreds of subtypes of motor neurons that differ in morphology, functional properties, gene expression patterns, molecular signatures, and connectivity. Little is known about how to identify/define terminal motor neuron subtypes or why some are more susceptible to disease processes than others. The goal of this research proposal is use a unique 'bottom-up' approach in C. elegans to identify defining factors and regulatory mechanisms for generating motor neuron subtypes and to understand the functional importance of motor neuron subtype diversity. This will be achieved by studying one cholinergic motor neuron subtype in the C. elegans ventral nerve cord, the AS motor neurons, and the potential role for unc-55 in specifying AS subtype fate. Using C. elegans and analysis of gene regulation, novel factors that molecularly define subtypes from one another will be identified, and the role for unc-55 in regulating these factors will be determined. Next, a candidate approach and an unbiased forward genetic screen will define the regulation of unc-55 in AS motor neurons, utilizing both manual and automated genetic screening techniques in C. elegans. Lastly, the functional importance of the AS motor neurons will be defined by combining optical ablation, optical neuronal activity monitoring, and optogenetic manipulation of AS motor neurons with advanced worm tracking and imaging in freely behaving worms. This proposal will define the specification and function of AS motor neurons, but also broadly address the mechanisms for generating motor neuron subtypes. Unraveling the molecular mechanisms that identify, regulate, and maintain motor neuron subtypes are particularly relevant to motor neuron degeneration, and may provide novel targets for pathogenesis, disease diagnosis, and therapeutics for motor neuron related diseases.